Reconstitution of the Entire Hepatitis C Virus Life Cycle inNonhepatic Cells
Daniel Da Costa,a,b Marine Turek,a,b Daniel J. Felmlee,a,b Erika Girardi,b,c Sébastien Pfeffer,b,c Gang Long,d Ralf Bartenschlager,d
Mirjam B. Zeisel,a,b and Thomas F. Baumerta,b,e
INSERM, U748, Strasbourg, Francea; Université de Strasbourg, Strasbourg, Franceb; Architecture et Réactivité de l’ARN, Institut de Biologie Moléculaire et Cellulaire duCNRS, Strasbourg, Francec; Department of Molecular Virology, University of Heidelberg, Heidelberg, Germanyd; and Pôle Hépato-digestif, Hôpitaux Universitaires deStrasbourg, Strasbourg, Francee
Hepatitis C virus (HCV) is a human hepatotropic virus, but the relevant host factors restricting HCV infection to hepatocytes areonly partially understood. We demonstrate that exogenous expression of defined host factors reconstituted the entire HCV lifecycle in human nonhepatic 293T cells. This study shows robust HCV entry, RNA replication, and production of infectious virusin human nonhepatic cells and highlights key host factors required for liver tropism of HCV.
Virus-host interactions that determine and restrict specific tis-sue and host tropisms have a complex evolutionary history
and also have significant consequences for the pathogenesis ofviral infection and human disease. Viral hepatitis is a major dis-ease burden. Indeed, infection of hepatocytes by a variety of hepa-totropic viruses from different orders and families can lead totissue inflammation, fibrosis, and hepatocellular carcinoma. Hep-atitis C virus (HCV), a member of the family Flaviviridae, is aprime example of a virus that causes chronic hepatitis worldwide.While HCV primarily infects hepatocytes of humans and chim-panzees, the virus has been shown to enter neuronal and endothe-lial cells of the blood-brain barrier. However, infection of thesecells occurs at a low level, and production of infectious viruses isgreatly diminished relative to that in liver-derived cells (9, 10).Unlike HCV, other members of the family Flaviviridae have amuch broader tissue and species tropism. For example, denguevirus infects and replicates both in the midgut epithelia of Aedesaegypti mosquitoes and in human monocytes and hepatocytes(20, 25, 39). Moreover, a virus closely related to HCV was recentlyidentified from respiratory samples from dogs (18). A large panelof host factors required for HCV has been identified so far (36).However, the key host factors mediating liver tropism of the virusand allowing reconstitution of the viral life cycle in human cellsare still only partially understood.
Taking advantage of our current knowledge of host factorsinvolved in HCV infection, we sought to engineer a human kidneycell line (293T) that would be capable of sustaining the entire HCVlife cycle. The aim was to define host factors that are necessary andsufficient for the HCV life cycle, in order to understand the livertissue specificity of HCV.
293T cells were obtained from ATCC and their identity wasverified by genomic profile comparison to the LGC Standardsdatabase by short tandem repeat profiling as described previously(1) (Fig. 1A). In order to render them infectible by HCV, we usedlentiviral vectors to express the four principal HCV host entryfactors— claudin-1 (CLDN1), CD81, occludin (OCLN), andscavenger receptor class B type I (SR-BI) (2, 7, 34, 35)— by usingpreviously described expression constructs and methods (3, 24).Four stable 293T cell lines were selected to express either CLDN1alone, CD81/OCLN with or without CLDN1, or CLDN1/CD81/OCLN together with SR-BI (293T-4R). After verifying stable ex-
pression of these proteins using receptor-specific antibodies (Fig.1B), we infected these cells with HCV pseudoparticles expressingthe envelope glycoproteins of HCV genotype 1b (HCVpp; HCV-Jstrain described in reference 31). While CLDN1 expression aloneconferred limited permissiveness for HCV infection, as previouslydescribed (7), expression of all four factors enhanced HCV entryto a level that was around 4-fold higher than that in Huh7.5.1 cells,which is the liver-derived model hepatoma cell line for studyingHCV infection (Fig. 1C).
Genuine cell culture infection of HCV (HCVcc) was then in-vestigated in 293T-4R cells using a chimeric virus composed oftwo genotype 2a isolates (designated Jc1 [19, 32]) and engineeredfor Renilla luciferase expression (JcR2a [38]). However, as shownin Fig. 2A, overcoming the HCV entry block was not sufficient forrobust viral RNA replication in 293T cells.
Several studies have shown that micro-RNA 122 (miR122) is aliver-specific host factor critical for HCV replication (5, 16, 17,28). Since Northern blot analyses demonstrated undetectablemiR122 expression in 293T-4R cells (Fig. 2C), we investigatedwhether exogenous miR122 expression reconstituted viral RNAreplication. Indeed, stable expression of this factor, by usingmiR122-encoding lentiviruses in the 293T-4R line, rendered thecells permissive for bona fide HCVcc infection, with replication tolevels comparable to those seen with Huh7.5.1 cells, as assessed byluciferase reporter activity (Fig. 2B). Further confirmation of gen-uine infection was obtained by observing similar infectivity (de-termined as 50% tissue culture infective doses [TCID50]) withHCVcc (Jc1) without a reporter gene, by detecting expression ofviral protein NS5A (Fig. 2B). We verified expression of miR122 intransduced 293T-4R/miR122 cells, and the level was comparable
to that in Huh7.5.1 cells, as assessed by Northern blotting (Fig.2C), and the cell proliferation rates of the different cell lines weresimilar (data not shown). Kinetics of HCV replication in 293T-4R/miR122 cells matched those of Huh7.5.1 cells, suggesting thataside from miR122, cell factors present in human liver- and kid-ney-derived cells are equally efficient for replication, as assayed byluciferase reporter gene expression (Fig. 2D). Expression of viralproteins in infected cells was further confirmed using HCV core-specific immunofluorescence (Fig. 2E) and flow cytometry (datanot shown).
To further confirm whether viral entry and replication in stablytransduced 293T cells are mediated by the same host and virusfactors as in human Huh7.5.1 hepatoma cells, we used well-char-acterized entry and replication inhibitors. Antibodies directedagainst the HCV entry factors CD81 (JS-81; BD Biosciences),CLDN1 (11), and SR-BI (M. N. Zahid, M. Turek, F. Xiao, V. L. D.Thi, M. Guérin, I. Fofana, P. Bachellier, J. Thompson, L. Delang, J.Neyts, D. Bankwitz, T. Pietschmann, M. Dreux, F.-L. Cosset, F.Grunert, T. F. Baumert, and M. B. Zeisel, submitted for publica-tion) were effective in inhibiting infection (Fig. 2F). Moreover,both a polyclonal serum recognizing Apolipoprotein E (ApoE)(29) and a monoclonal antibody recognizing the low-density-li-poprotein (LDL) receptor binding domain of ApoE (37) effec-tively neutralized HCV infection of 293T-4R/miR122 cells (Fig.2F). The same was true for the recently identified HCV entry in-hibitor erlotinib, which targets the kinase activity of the host entryregulatory protein, epidermal growth factor receptor (EGFR)(Fig. 2F) (24). Likewise, the well-characterized inhibitors of HCVNS3 protease and polymerase telaprevir (VX950) and mericit-abine (R7128) impaired HCV replication in 293T-4R/miR122cells (Fig. 2F). These data demonstrate that HCVcc RNA replica-tion in kidney-derived 293T-4R/miR122 cells is efficient and de-pendent on mechanisms similar to those in liver-derivedHuh7.5.1 cells.
Despite efficient entry and RNA replication of 293T-4R/miR122 cells infected with recombinant HCVcc, these cells didnot release infectious virions, suggesting that kidney-derived cellslack factors required for viral assembly and release. Therefore, weaimed to reconstitute virus production by expression of HCV as-sembly factors. HCV production shares factors involved in very-low-density lipoprotein (VLDL) assembly, a process that occursexclusively in hepatocytes (13, 14, 27). While the necessity of apo-lipoprotein B (ApoB) in HCV production is controversial (15),ApoE is known to be critical and is incorporated into the virion(26). We therefore expressed the most common isoform of ApoE(ApoE3) in 293T-4R/miR122 cells by using a lentiviral vector en-coding human ApoE3 as described previously (23) and confirmedits expression by flow cytometry using an ApoE-specific antibody(Fig. 3A). We then infected 293T-4R/miR122/ApoE cells. Subse-quently, the production and release of viral particles was assessedby incubating naïve Huh7.5.1 cells with the supernatants fromthese cells. Indeed, 293T-4R/miR122/ApoE released infectious
FIG 1 Expression of four HCV entry factors renders 293T cells highly permis-sive to HCVpp entry. (A) Short tandem repeat (STR) profile of the 293T cellsused in this study (cell line authentication, LGC Standards) was performed asdescribed previously (1). The names of tested loci are in bold, and peak posi-tions from STR profile of 293T cells were compared to the LGC Standardsdatabase. (B) 293T cells (cultured in Dulbecco’s modified Eagle medium withhigh glucose; Life Technologies) were transduced with lentiviruses (as de-scribed in reference 3) to express given HCV entry factors. After transduction,cells were selected with 12 �g/ml of blasticidin for 2 weeks. Blasticidin-resis-tant cells were assessed by flow cytometry using monoclonal antibodies(CLDN1 [11], OCLN [catalog no. 33–1500; Invitrogen], and SR-BI [Zahid etal., submitted for publication]) recognizing indicated entry factors. Entry fac-tor-transduced cells (dark gray histograms) were compared to naïve 293T cells(light gray histograms) and isotype control antibody (catalog no. 10400C; LifeTechnologies) (white histograms with dashed lines). The x axis shows fluores-cence intensity; the y axis shows the number of events. (C) Transduced 293Tcells were assessed for HCVpp (genotype 1b; HCV-J strain; produced as de-scribed in reference 31) entry by determining luciferase activity 72 h postin-fection as previously described (35). Results were first normalized to vesicular
stomatitis virus pseudoparticle entry (VSV-Gpp; produced as described inreference 8) and then compared to those obtained with Huh7.5.1 cells (cul-tured as described in reference 41). Results are means and standard deviations(SD) from three independent experiments performed in triplicate. Entry isrelative to entry into Huh7.5.1 cells, and 100% relative infectivity is repre-sented by a solid line. Statistical analysis for entry factor expressing cells rela-tive to naïve 293T cells was performed using the Student t test (*, P � 0.05).
FIG 2 293T-4R cells support robust HCV infection upon miR122 expression. (A) Stable 293T-4R cells described in the legend to Fig. 1 were challenged withHCVcc (JcR2a; produced as described in reference 38) or were mock infected, and luciferase activity was assessed 72 h postinfection as described previously (38).Results are means and SD, in relative light units (RLU), from three independent experiments performed in triplicate. (B) 293T-4R cells were stably transducedusing miR122-encoding lentiviruses (catalog no. mh15049; ABM) and 2.5 �g/ml of puromycin-resistant cells were selected over 2 weeks. 293T-4R/miR122 cellsand Huh7.5.1 cells were then infected with HCVcc or mock infected for 6 h. Infection was assayed by monitoring luciferase activity 72 h postinfection. Resultsare means and SD from three independent experiments performed in triplicate. Jc1, an HCVcc without a luciferase reporter (32), was likewise used to infectHuh7.5.1 and 293T-4R/miR122 cells, and its infectivity was assessed by limiting-dilution assay (TCID50) by detecting viral protein NS5A using immunohisto-chemistry, represented as gray bars (22). Results are expressed as means and SD from three independent experiments. (C) Northern blots of miR122 and miR-16,and U6 RNA as a loading control, extracted from 293T-4R cells, 293T-4R cells stably expressing miR122, and Huh7.5.1 cells as positive control. Northern blottingusing a miR122-specific probe were performed as described previously (30). Oligonucleotide lengths (in nucleotides [nt]) are indicated on the left. (D) 293T-4R,293T-4R/miR122, and Huh7.5.1 cells were incubated side by side with HCVcc (JcR2a), and luciferase activity was monitored every 24 h over a 72-h period.Results are means and SD from three independent experiments performed in triplicate. (E) Huh7.5.1, 293T-4R, and 293T-4R/miR122 cells were infected for 72h, and HCV core protein (core antibody C7-50; Thermo Scientific), or nonspecific IgG as a control (catalog no. 10400C; Life Technologies), was observed byimmunofluorescence; nuclei were stained using DAPI (4=,6=-diamidino-2-phenylindole). (F) 293T-4R/miR122 cells were preincubated for 1 h at 37°C with theindicated entry inhibitors, antivirals, or controls (20 �g/ml of monoclonal antibodies [MAb], anti-CD81 [JS81; BD Biosciences], anti-CLDN1 [11], andanti-SR-BI [Zahid et al., submitted]; 1:200 dilution of polyclonal antibody [PAb] anti-ApoE [catalog no. 178479; Calbiochem]; 20 �g/ml anti-ApoE MAb [37];10 �M erlotinib [catalog no. E-4997; LC Laboratories]; 1 �M protease inhibitor telaprevir VX950; 1 �M polymerase inhibitor mericitabine R7128 [bothsynthesized by Acme Bioscience Inc.]; and 0.7% dimethyl sulfoxide [DMSO]) and then infected with HCVcc (JcR2a) in the presence of the given entry inhibitorsor antivirals. Cell lysates were assessed for luciferase activity 72 h postinfection. Results are means and standard errors of the means from three independentexperiments performed in triplicate. Values are relative to controls, and 100% relative infectivity is represented by a solid line. In panels A, B, and D, detectionlimits are represented by dashed lines. Statistical analysis relative to control was performed using the Student t test (*, P � 0.05).
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HCV particles as shown by a marked and highly significant in-crease in infectivity (as assessed by luciferase activity of JcR2a virusand TCID50 of Jc1 virus without a reporter gene) of the superna-tant compared to the supernatant of 293T-4R/miR122 cells with-out ApoE expression (Fig. 3B). Although the production of infec-tious particles was lower than in Huh7.5.1 cells studied in side-by-side experiments, these data indicate that ApoE is a key factor forvirus production in reconstituting the viral life cycle in nonhepaticcells. This diminished HCV production was not due to dimin-ished replication levels, as ApoE-transduced cells had HCV repli-cation levels similar to those of 293T-4R/miR122 cells prior toApoE expression (data not shown). To test if HCV produced bythese cells is reliant only on the human ApoE3 isoform or coulduse other forms of ApoE, we similarly transduced human ApoE2and ApoE4 isoforms, as well as murine ApoE (Fig. 3C). Virusesproduced from 293T cells expressing these ApoE isoforms and themouse ortholog had similar infectivity compared to humanApoE3 isoform (Fig. 3D).
Focusing on the most common ApoE isoform (ApoE3), we
further characterized the kinetics and attributes of these viruses.First, we confirmed that HCV particles from engineered 293T cellscould establish infection by monitoring the increase in HCV ge-nomes over time in Huh7.5.1 target cells after exposure to thesupernatant of HCVcc-infected 293T-4R/miR122/ApoE cells(Fig. 4A). Next, we characterized the kinetics of HCV RNA pro-duction from infected 293T-4R/miR122/ApoE cells by measuringHCV RNA in the media at serial time points following infection(Fig. 4B). Interestingly, the levels of HCV RNA released into theculture media of 293T-4R/miR122/ApoE cells was similar to levelsof HCV RNA in the media of Huh7.5.1 cells after 72 h, whereascells that were not transduced with ApoE released minimalamounts of HCV RNA, likely due to previously reported nonspe-cific release of HCV RNA during replication (Fig. 4B) (33). Thesedata suggest that the specific infectivity differs between virus pro-duced from Huh7.5.1 cells and 293T cells engineered to expressessential host factors. An estimation of the specific infectivity ofthe released viruses (TCID50/HCV RNA genomes) revealed ap-proximately a 30-fold difference between the differently derived
FIG 3 Infectious HCV particles are released from 293T-4R/miR122 cells upon ApoE expression. (A) 293T-4R/miR122 cells were transduced with an ApoE3-encoding lentiviral vector described in reference 23. At 72 h posttransduction, both transduced and untransduced cells were stained for flow cytometry analysis.ApoE expression was analyzed using a specific ApoE antibody (clone D6E10, catalog no. ab1906; Abcam) (untransduced cells are represented as light grayhistograms, and transduced cells are shown by dark gray histograms), and an isotype antibody (catalog no. 10400C; Life Technologies) was used as a control(white histograms with dashed lines). Huh7.5.1 cells were used for control of ApoE expression, and PBS is the control for the isotype antibody (histogram withthe thick black outline). (B) Transduced 293T-4R/miR122/ApoE cells were infected with HCVcc (JcR2a or Jc1). At 6 h postinfection, cells were washed threetimes with PBS, and fresh culture medium was added. At 72 h postinfection, medium from infected cells was passaged onto naïve Huh7.5.1 cells. Lysates ofJcR2a-infected cells were assessed for luciferase activity 72 h postinfection. Results are means and SD from three independent experiments performed in triplicate.The detection limit is represented by a dashed line. The infectivities of Jc1 derived from infected Huh7.5.1 or 293T-4R/miR122/ApoE cells were assessed bylimiting-dilution assay (TCID50) by detecting NS5A by immunohistochemistry, represented as gray bars. Results are means and SD from three independentexperiments. #, levels were below the limit of detection. Statistical analysis relative to the control was performed using the Student t test (*, P � 0.05). (C)293T-4R/miR122 cells were transduced with the indicated ApoE isoform-encoding lentiviral vectors (24) or mock transduced (control). At 72 h posttransduc-tion, cells were either lysed or seeded for HCVcc infection. Cell lysates were assessed for ApoE expression by Western blotting either by using ApoE antibody(clone D6E10, catalog no. ab1906; Abcam) for human ApoE (h-ApoE) expression or by using a mouse ApoE-specific antibody for mouse ApoE (m-ApoE)expression (catalog no. ab20874; Abcam). Huh7.5.1 and primary mouse hepatocytes (PMH) were used as controls for human and mouse ApoE expression,respectively. (D) The different ApoE isoform-expressing 293T-derived cells were assessed for their capacity to produce infectious virus by infecting them withHCVcc (JcR2a), and 72 h postinfection, supernatants of infected 293T-derived cells were passaged onto naïve Huh7.5.1 cells. At 72 h after infection was initiated,Huh7.5.1 cells were lysed and luciferase activity assessed. Results are means and SD from a representative experiment performed in triplicate. The dashed linerepresents the detection limit.
viruses (1/900 for Huh7.5.1-derived virus and 1/26,000 for 293T-4R/miR122/ApoE-derived virus). It should be noted that HCVparticles produced from 293T-4R/miR122/ApoE cells proved tohave a route of infection similar to that of liver-derived HCVcc, inthat entry into Huh7.5.1 cells was neutralized by well-character-ized HCV entry inhibitors, including CD81-, SR-BI-, CLDN1-,and ApoE-specific antibodies and erlotinib (Fig. 4C). Fractionat-ing the virus by iodixanol density gradients revealed that the in-fectious virions produced from 293T-4R/miR122/ApoE cells havea buoyant density similar to those from Huh7.5.1 cells (Fig. 4D).
The data presented here demonstrate that trans-expression ofOCLN, CD81, CLDN1, SR-BI, miR122, and ApoE endows 293Thuman kidney-derived cells with the capacity to support the com-plete HCV life cycle. Expression of four principal entry factors andmiR122 generated cells with higher entry levels than and similarreplication kinetics to those of the extensively optimized Huh7.5.1cells (4, 41). It should be noted in this context that the recentlyidentified entry factor EGFR is also expressed in 293T cells (datanot shown) (24, 40). We confirmed that expression of CLDN1alone appears to be sufficient for infection of 293T cells (7) andexpand these findings by showing that high-level expression of thefour canonical HCV entry factors makes previously impenetrablecells fourfold more permissive than Huh7.5.1 cells. These obser-
vations were confirmed by HCVcc infection of 293T cells engi-neered to express miR122 in addition to variable sets of entryfactors (data not shown). While the present study focused on en-gineering a human cell line for infection, it has been demonstratedthat concomitant high-level expression of the four human entryfactors is required for robust HCV entry into mouse hepatocytesin vivo (6). Since none of the identified entry factors are exclusivelyexpressed in the liver, it is likely that the combined expression ofthese host factors at substantial levels allows the virus to produc-tively infect the human liver, rather than a single liver-specificentry factor restricting HCV infection.
Investigators have shown that miR122 expression increasesHCV replication in mouse embryonic fibroblasts and other hep-atoma cell lines such as HepG2 cells (17, 21, 28). Furthermore,HEK-293 cells modified to express miR122 are capable of sustain-ing selectable HCV subgenomic replicons, although expression ofmutated miR122, at sites required for HCV RNA binding, can alsosustain these replicons (5). We demonstrate here de novo replica-tion following an infection event of a nonhepatic cell line engi-neered to express HCV host factors. Our data also demonstratethat there is no restrictive factor of HCV entry and viral RNAreplication that is present in 293T cells. HCV entry and replicationin human blood brain barrier endothelial and neuronal cells have
FIG 4 Characterization of HCVcc derived from 293T-4R/miR122/ApoE cells. (A) Culture media from Jc1-infected 293T-4R/miR122, 293T-4R/miR122/ApoE,and Huh7.5.1 cells were passaged onto naïve Huh7.5.1 target cells. Total RNA from these Huh7.5.1 target cells was extracted at the indicated time points, andHCV RNA was quantitated by reverse transcription-quantitative PCR (RT-qPCR) as described previously (11). Values were normalized to the value for theinternal control gene (GAPDH gene). Results are means and SD from an experiment performed in quadruplicate. (B) HCV RNA production was measured byinfecting 293T-4R/miR122, 293T-4R/miR122/ApoE, and Huh7.5.1 cells side by side with HCVcc (Jc1). RNA from supernatants of infected cells was extracted atthe indicated time points, and HCV RNA was quantitated by RT-qPCR. Results are means and SD from an experiment performed in triplicate. (C) Culture mediaof infected 293T-4R/miR122/ApoE cells were harvested 72 h postinfection and passaged onto naïve Huh7.5.1 cells that had been preincubated with either controlIgG, DMSO, or the indicated entry inhibitors. Results are mean percentages of HCV infection (as assessed by luciferase activity) relative to the control and SDfrom a representative of two independent experiments performed in triplicate, and 100% relative infectivity is represented by a solid line. The virus used wasJcR2a with a TCID50 of 105 to 106/ml. (D) Density distributions of infectious 293T-4R/miR122/ApoE- and Huh7.5.1-derived HCVcc (Jc1) were determined byoverlaying 0.5 ml of culture medium on a 5-ml, 4-to-40% iodixanol step gradient and ultracentrifuging samples for 16 h at 40,000 rpm on an SW-55 rotor(Beckman Coulter). Fractions were carefully harvested from the top of each tube, and density was determined by weighing 0.5 ml of each fraction. Each fractionwas assayed for infectivity by TCID50 by detecting NS5A as described previously (22).
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been described (9, 10). In contrast to the kidney-derived cells de-scribed here, HCV replication in blood-brain barrier endothelialcells occurred via a miR122-independent mechanism but at a di-minished level (9). Thus, the cell lines developed in this study maybe useful as a tool to further understand the molecular mecha-nisms of extrahepatic infection.
The production of HCV in 293T-4R/miR122/ApoE cells wasdiminished relative to that in Huh7.5.1 cells but markedly andsignificantly higher than that in cells without ApoE expression.This demonstrates that apart from ApoE, all the other factors nec-essary for the production of infectious particles are present in293T cells, yet additional host factors may increase efficient pro-duction levels. The cell line generated in this study is likely to allowfurther discovery of the minimal set of host factors required forrobust viral production. Additional relevant factors enhancing vi-ral production may be ApoB (27), DGAT1 (13), or microsomaltriglyceride transfer protein (MTP) (12, 14). Notably, ApoE hasrecently been demonstrated to be essential for virus production;ApoE-deficient mouse hepatocytes with trans expression of HCVRNA and proteins along with ApoE are able to produce high levelsof infectious virions (23).
In summary, this study demonstrates that a small set of definedhost factors is sufficient to reconstitute the complete viral life cyclein nonhepatic cells. These results advance our knowledge of tis-sue-specific factors for HCV infection and provide novel tools toelucidate host and restriction factors for the HCV life cycle.
ACKNOWLEDGMENTS
This work was supported by the European Union (ERC-2008-AdG-233130-HEPCENT, INTERREG-IV-Rhin Supérieur-FEDER-Hepato-Regio-Net 2009), an EASL fellowship to D.J.F., ANRS (2011/132), theLaboratoire d’Excellence HEPSYS (Investissement d’Avenir; ANR-10-LAB-28), an ANRS fellowship to E.G., INSERM, CNRS, and the Univer-sité de Strasbourg.
We thank T. Pietschmann (Division of Experimental Virology,TWINCORE, Hannover, Germany) for providing the lentiviral vectorsencoding HCV entry factors, F.-L. Cosset for providing plasmids for theproduction of HCVpp, D. Trono (Ecole Polytechnique Fédérale de Lau-sanne, Switzerland) for pWPI plasmid, R. Milne for monoclonal ApoEantibody, M. Harris for HCV NS5A antibody used for immunohisto-chemistry, and F. Chisari for Huh7.5.1 cells. We acknowledge Sarah Du-rand (INSERM U748, Strasbourg) and Charlotte Bach (INSERM U748,Strasbourg) for excellent technical work. We are thankful to Heidi Barth(INSERM U748, Strasbourg) and Catherine Schuster (INSERM U748,Strasbourg) for helpful discussions.
M.B.Z. and T.F.B. designed and supervised the research. D.D.C., M.T.,D.J.F., E.G., S.P., G.L., R.B., M.B.Z., and T.F.B. performed research.D.D.C., M.T., E.G., S.P., M.B.Z., and T.F.B. analyzed data. R.B. providedimportant ideas for the initiation and execution of this study and providedreagents. D.D.C., M.T., M.B.Z., D.J.F., and T.F.B. wrote the manuscript.
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